Extracellular vesicle‐carried microRNA‐27b derived from mesenchymal stem cells accelerates cutaneous wound healing via E3 ubiquitin ligase ITCH

Abstract Mesenchymal stem cells (MSCs) have been highlighted as promising candidate cells in relation to cutaneous wound healing. The current study aimed to investigate whether MSC‐derived extracellular vesicles (EVs) could transfer microRNA‐27b (miR‐27b) to influence cutaneous wound healing. The miR‐27b expression was examined in the established cutaneous wound mouse model, and its correlation with the wound healing rate was evaluated by Pearson's correlation analysis. The identified human umbilical cord MSC‐derived EVs were co‐cultured with human immortal keratinocyte line HaCaT and human skin fibroblasts (HSFs). The mice with cutaneous wound received injections of MSC‐derived EVs. The effects of EVs or miR‐27b loaded on wound healing and cellular functions were analysed via gain‐ and loss‐of‐function approaches in the co‐culture system. Dual‐luciferase reporter gene assay was employed to verify the relationship between miR‐27b and Itchy E3 ubiquitin protein ligase (ITCH). Rescue experiments were conducted to investigate the underlying mechanisms associated with the ITCH/JUNB/inositol‐requiring enzyme 1α (IRE1α) axis. miR‐27b was down‐regulated in the mouse model, with its expression found to be positively correlated with the wound healing rate. Abundant miR‐27b was detected in the MSC‐derived EVs, while EV‐transferred miR‐27b improved cutaneous wound healing in mice and improved proliferation and migration of HaCaT cells and HSFs in vitro. As a target of miR‐27b, ITCH was found to repress cell proliferation and migration. ITCH enhanced the JUNB ubiquitination and degradation, ultimately inhibiting JUNB and IRE1α expressions and restraining wound healing. Collectively, MSC‐derived EVs transferring miR‐27b can promote cutaneous wound healing via ITCH/JUNB/IRE1α signalling, providing insight with clinical implications.

wound healing. 12 It has been highlighted that the transformation of anti-inflammatory RNAs from stem cells to injury sites mediated by EVs could coordinate the inflammatory responses and immune alleviation, thereby better facilitating the healing processes, which exerts a value and potential for the therapeutic approaches in regenerative medicine. 13 Accumulating studies have suggested EVs as novel regulators of cell-to-cell communication. 14 EVs derived from MSCs serve as a potential cell-free therapy for cutaneous regeneration and wound healing. 15 EVs encompass proteins, messenger RNAs (mRNAs) and miRNAs, which can transfer between cells. 16 miRNAs have been revealed to associate with the event of pathologic wound healing and hypertrophic scar formation. 17 Additionally, miR-27b has been recently identified as a target miRNA that helps to accelerate the process of wound healing in type 2 diabetic mice. 18 The interaction between miRNAs and their specific target genes has been proposed to be a potential biomarker and therapeutic target of human diseases. 19,20 The starBase (tarbase.sysu.edu.cn/) predicted binding sites between miR-27b and the 3'-untranslated region (UTR) of Itchy E3 ubiquitin protein ligase (ITCH) mRNA. ITCH represents a ubiquitin E3 ligase that regulates protein stability, which has been previously documented to confer protection against the progression of autoimmune disease in human and mice. 21 Existing literature has suggested that ITCH functions in epidermal homeostasis and remodelling. 22 Of note, JUNB (a member of the AP-1 transcription factor family) can define functional and structural integrity of cutaneous epidermo-pilosebaceous unit. 23 The JUNB neddylation and JUNBdependent transcription are modulated by ITCH. 24 Moreover, JUNB has been shown to increase the expression of inositol-requiring enzyme 1α (IRE1α) in osteoblastogenesis, 25 while IRE1α has been reported to accelerate the process of wound healing in diabetes. 26 Following the aforementioned exploration of literature, we asserted the hypothesis that miR-27b from MSC-derived EVs could influence cutaneous wound healing via regulation of ITCH. Hence, our study was designed to validate this hypothesis and to elucidate the ITCH/ JUNB/IRE1α axis.

| Identification of HUCMSCs in vitro
The HUCMSCs were seeded in 6-well plates at a density of 1 × 10 5 cells/well. After attachment, the cells were cultured with an osteogenic medium containing DMEM, 0.17 mmol/L vitamin C, 0.5% FBS, 10 mmol/L β-glycerophosphate, 100 nmol/L dexamethasone and 1% penicillin/streptomycin (Sigma, St Louis, MO) over a period of 21-28 days with the medium changed every 2 days. Once calcium nodules were visualized under a light microscope (Leica, Frankfurt, Germany), the cells were stained with Alizarin red S staining for further analysis.
HUCMSCs were seeded into 15-mL centrifuge tubes with a density of about 2 × 10 6 cells/tube and cultured at 37°C with 5% CO 2 for 24 hours. After 24 hours, the cells were cultured in chondrogenic medium containing DMEM (4.5 g/L glucose) supplemented with 100 nmol/L dexamethasone, 0.35 mmol/L proline, 0.17 mmol/L vitamin C, 1 mmol/L sodium pyruvate, 1% insulin-transferrin-selenium, 10 ng/mL TGFβ-3 and 1% penicillin/streptomycin (Sigma) for 21-28 days at 37°C with 5% CO 2 , after which the medium was replaced with a fresh medium on the following day. After the cells had grew into cell spheres with a diameter of 1.5-2.0 mm, the cells were sliced into sections, stained by Alcian blue, and observed under the light microscope.

| Extraction of HUCMSC-derived EVs
The HUCMSCs were subsequently seeded into a 6-well plate at a density of 1 × 10 5 cells/well and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (Gibco, Grand Island, NY) until cell confluence reached 80%. The cells were then cultured for 48 hours with Roswell Park Memorial Institute (RPMI) 1640 medium containing EV-depleted FBS (centrifuged at 100 000 g for 18 hours).
The cell supernatant was then centrifuged using Beckman Avanti Centrifuge J-26XP (Beckman Coulter) for debris and apoptotic body removal. The supernatant was subsequently centrifuged at 110 000 g for 70 minutes (Beckman Optima L-80 XP Ultracentrifuge with 70Ti rotor), followed by purification by centrifugation at 110 000 g for 70 minutes. All the centrifugations were conducted at 4°C. The precipitates were then resuspended in PBS and sterilized by filtration through a 0.22-μm filter (Millipore, Darmstadt, Germany).

| Identification of HUCMSC-derived EVs
HUCMSC-derived EVs were measured using Nanosizer™ instrument 'dynamic light scattering (DLS)' (Malvern Instruments, Malvern, UK). The EVs were diluted in 1 mL of PBS and mixed well. The diluted EVs were subsequently injected into the NanoSight NS300 instrument with the particles automatically tracked and sized using Nanoparticle Tracking Analysis (NTA). The morphology of the EVs was observed and analysed using a Hitachi H-7500 transmission electron microscope (TEM; Hitachi, Tokyo, Japan). More specifically, 10 μL of EVs was placed on formvar carbon-coated 200-mesh copper electron microscopy grids, incubated for 5 minutes and stained using standard 1% uranyl acetate for 1 minutes. Prior to TEM observation, the grids were washed three times with PBS and semi-dried.

| Establishment of mouse cutaneous wound model
A total of 90 Kunming male mice aged 9-12 weeks (weight of 26-30 g) were recruited in this study. The mice were subsequently anaesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium (Sigma) prior to surgical experimentation. Two full-thickness excisional cutaneous wounds (with a diameter of 12 mm) were made on the dorsum of mice. Following successful model establishment, 30 mice were randomly selected in order to identify the relationship between wound reduction rate and miR-27b expression.
On the fourth day, the wound tissue was photographed with the reduction rate calculated, after which the skin tissue was extracted to determine the expression of miR-27b. The remaining mice were randomly classified into 4 groups (15 mice per group). Mice in the experimental group were subcutaneously injected with MSCs-EVs (200 μg dissolved in 100 μL PBS), MSCs-EVs-inhibitor-NC, MSCs-EVs-miR-27b-inhibitor or an equal volume of PBS at 4 injection sites around the vicinity of the wound. On the 0, 2, 4, 6, and 8 days after operation, these mice were subjected to wound photographing and recording, respectively. All wounds were measured using vernier calliper with the wound area evaluated using Image-Pro Plus 6 software (Media Cybernetics, Bethesda). The wound reduction rate was calculated using the following formula: wound reduction rate (%) = (A0-At)/A0 × 100, where A0 was indicative of the initial wound area, and At represented the wound area at 2, 4, 6 and 8 days after the operation. Fifty per cent of the wound healing rate was regarded as the threshold, with a rate above 50% considered to indicate a high healing ability, while <50% was viewed as low healing ability. At 8 days after operation, the mice were killed with their skin samples harvested. The skin samples were subject to histopathological and molecular analyses.

| Histopathological analyses
The wound bed and surrounding healthy skin of the mice were collected for further experiments. Haematoxylin-eosin (HE) staining was performed after which the wounds and surrounding skins (4 mm 2 ) were fixed by 4% paraformaldehyde (pH = 7.4), dehydrated by gradient alcohol, embedded in paraffin, and sliced into 4-μm sections, followed by HE staining (Beyotime) and observation under a light microscope. Based on a previously reported method, 28 Masson's trichrome staining was conducted to evaluate re-epitheli-

| Internalization of MSC-derived EVs by HSFs and HaCaT cells
Next, in order to determine the internalization of MSC-derived EVs by HSFs and HaCaT cells, 10 μg of EVs was labelled using green fluorescent dye (PKH67; Sigma) and incubated with 1 × 10 5 cells at 37°C for 3 hours. The cells were then fixed with 4% paraformaldehyde for 15 minutes followed by the addition of 4',6-diamidino-2-phenylindole (DAPI) (0.5 mg/mL; Invitrogen) for nucleus staining. Finally, green fluorescence was observed under a fluorescence microscope (Leica DMI6000B, Solms, Germany).
Next, to investigate the internalization of MSC-derived EVs carrying miR-27b by HSFs and HaCaT cells, Lipofectamine 3000 reagent (L3000001, Invitrogen) was employed to transfect the cy3-conjugated miR-27b (GenePharma, Shanghai, China) into MSCs in a serum-free medium. After 6 hours, the medium was renewed using a medium containing 10% serum without EVs for further incubation for 48 hours. The cell supernatant was then collected, centrifuged and resuspended using PBS, which was then added into the HSFs and HaCaT cells, respectively. Finally, the cells were fixed with 4% paraformaldehyde for 15 minutes and added with DAPI (0.5 mg/ mL; Invitrogen) for nucleus staining, followed by fluorescence microscopic observation (Leica DMI6000B, Solms, Germany).

| Cell counting kit-8 (CCK-8) assay
CCK-8 assay (Dojindo, Kyushu Island, Japan) was applied to detect cell proliferation. The cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well. At 1-5 days after treatment, 100 μl of fresh medium containing 10 μl of the CCK-8 solution was added to each well and incubated at 37°C for 1 hour. A Bio-Rad 680 microplate reader (Bio-Rad, Hercules, CA) was used to measure the absorbance at a wavelength of 450 nm. Cell proliferation was expressed as follows: cell proliferation = the absorbance of experimental wells -the absorbance of blank wells.

| Dual-luciferase reporter gene assay
The human ITCH 3'-UTR sequence or the mutant sequence of ITCH 3'-UTR containing the predicted miR-27b binding sites was inserted into the pGL3 promoter vector (GenScript, Nanjing, China).
The HEK293T cells (ATCC, Maryland) were then seeded into 24well plates at a density of 5 × 10 5 cells/well on the day prior to transfection. Next, the cells were co-transfected with luciferase reporter vectors (0.12 μg) and miR-27b mimic or mimic NC using Lipofectamine 3000 reagents (Invitrogen).

| RNA isolation and quantitation
TRIzol reagents were employed to extract the total RNA (Invitrogen).
Next, 1 µg total RNA was reverse transcribed into complementary DNA (cDNA) using the Revert Aid first-strand cDNA synthesis kit (Fermentas, Life Sciences, Canada). The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted using a SYBR Premix ExTaqTMII kit in an ABI PRISM® 7900HT System (Takara, Tokyo, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regarded as an internal reference, with the relative mRNA expression determined using the 2 −ΔΔCt method. The primer sequences used are depicted in Table 1 with the reverse primers provided by the TaqMan microRNA assay kit. The level of mRNA was normalized to the U6 gene.

| Western blot analysis
The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride membranes (Immobilon-P, Millipore, Billerica, MA, USA). The membranes were then blocked with 5% milk and Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hours, followed by incubation with primary antibodies at 4°C overnight. The next day, the membranes were re-probed with horseradish peroxidase (HRP)- Image-Pro Plus 6.0 software was employed for quantitative analysis of protein band intensity, and the relative expression of proteins was expressed as the ratio of the band intensity of target protein to that of GAPDH (A0280, 1:2000, rabbit antibody, abclonal). Abbreviations: RT-qPCR, reverse transcription quantitative polymerase chain reaction; miR-27b, microRNA-27b; ITCH, Itchy E3 ubiquitin protein ligase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRE1α, inositol-requiring enzyme 1α; F, forward, R, reverse.

| Statistical analysis
Data analysis was performed using SPSS 21.0 statistical software (IBM Corp. Armonk, NY). The measurement data were expressed as mean ± standard deviation. Unpaired data that conformed to normal distribution and homogeneity of variance between two groups were compared by unpaired t test. Data among multiple groups were compared by one-way analysis of variance (ANOVA). The correlation between the two parameters was analysed using Pearson's correlation analysis. A value of P < .05 was considered to be reflective of statistical significance.

| miR-27b exhibits poor expression in the cutaneous wound tissues of mice with low healing ability
As per a previously conducted study, miR-27b is associated with wound healing in type 2 diabetes mellitus. 18 Next, to determine the relationship between miR-27b expression and cutaneous wound healing, full-thickness excisional skin wounds were exerted on the dorsum of mice. On day 4th post-wounding, the wound area was measured followed by collection of region of wound skin, with RT-qPCR subsequently performed to determine the expression of miR-27b. As illustrated in Figure 1A, the expression of miR-27b was higher in the cutaneous wound tissues of mice with high healing ability than that of the mice with low healing ability.
Moreover, Pearson's correlation analysis ( Figure 1B) suggested that the wound reduction rate was positively correlated with miR-27b expression in cutaneous wound tissues. Therefore, miR-27b expressed poorly in the cutaneous wound tissues of mice with low healing ability and its expression was positively associated with wound healing.

| miR-27b is enriched in HUCMSC-derived EVs
HUCMSC-derived EVs have been reported to accelerate the rate of healing of scalded cutaneous wounds in rats, 30 and thus, we speculated that HUCMSC-derived EVs could improve wound healing by secreting miR-27b. In order to verify this hypothesis, The results of RT-qPCR in Figure 2I

| MSC-derived EVs carrying miR-27b potentiates proliferation and migration of HaCaT cells and HSFs in vitro
Next, to investigate the finer mechanisms associated with MSC-

| ITCH is targeted by EV-carried miR-27b in HaCaT cells and HSFs
Next, we set out to identify the downstream regulatory mechanism of miR-27b, the starBase (tarbase.sysu.edu.cn/), microRNA. www.targe tscan.org/vert_71/) were employed to predict the downstream target genes of miR-27b. Next, the predicted genes from the five databases were intersected ( Figure 4A), which revealed 502 candidate target genes (Table S1). The target genes were further subjected to gene interaction analysis using the STRING database (https://string-db.org/), with a gene interaction network map ( Figure 4B) subsequently constructed. The top 10 genes with the highest core degree were calculated ( Figure 4C). At the same time, the KOBAS3.0 database (http://kobas.cbi.pku.edu.cn/kobas 3/?t=1) was explored for KEGG pathway enrichment analysis ( Figure 4D).
Analysis using protein-protein interaction (PPI) network and KEGG suggested that ITCH was not only at the core of gene interaction network, but also significantly enriched in the TNF signalling pathway, which has been proposed to be involved in the inter-vertebral disc disease (IVDD) regulation. 31 Next, the starBase database used to predict the target gene of miR-27b with a binding site between miR-27b and ITCH detected ( Figure 4E). Dual-luciferase reporter gene assay provided evidence indicating that the luciferase activity of

| The transfer of miR-27b by MSC-derived EVs enhances proliferation and migration of HaCaT cells and HSFs through suppression of ITCH in vitro
The

| MSC-derived EVs carrying miR-27b facilitates proliferation and migration of HaCaT cells and HSFs via enhancement of the JUNB/IRE1α axis by inhibiting ITCH both in vitro and in vivo
Based on aforementioned findings, we hypothesized that miR-

| MSC-derived EVs carrying miR-27b enhances epidermal re-epithelialization and collagen fibre proliferation, thus advancing cutaneous wound healing in vivo
In order to further elucidate the effects associated with MSC-   Figure 7A) with the wound healing rate calculated ( Figure 7B).  Figure 7C) revealed that treatment with EVs derived from MSCs transfected with inhibitor-NC led to the formation of more new epidermis and dermis compared with PBS treatment. As illustrated in Figure 7D  ITCH has also been highlighted to exert a crucial effect on posttranslational modification through ubiquitin proteasomal protein degradation. 45 Our results illustrated that ITCH degraded JUNB through the ubiquitination and reduction of JUNB expression. ITCH depletion can enhance the cutaneous wound healing, and JUNB has been emphasized in previous reports as a crucial ITCH's substrate associated with epidermal development and homeostasis. 22 JUNB also elevates the expression of IRE1α in osteoblastogenesis, 25 and IRE1α promotes the wound healing in diabetes. 26 Furthermore, IRE1α overexpression in bone marrow-derived progenitor cells has been demonstrated to accelerate wound healing in diabetic settings. 46 Our study provided evidence to support the observation that EV-encapsulated miR-27b However, the research is still at the preclinical stage. A transition of this finding to clinical application warrants further investigation with the specific mechanism requiring further elucidation in future studies.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. Extensive efforts were made to ensure minimal suffering of the included animals.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Proliferation Migration